In microscopy, adequate and appropriate illumination of the object to be imaged by a microscope is essential. There must be enough light provided to the object to permit a viewer or detector to discern features of the object in the image produced by the microscope. In addition, the manner in which the light is provided to the object makes a difference in what features can be detected and the contrast with which they are imaged. Fundamentally, if the object to be imaged is transparent, it can be illuminated so that light passes through it and is modulated by the features of the object. This type of illumination is known as dia-illumination, through illumination or trans-illumination. On the other hand, if the object is opaque, it must be illuminated so that the light used to form an image of the object is emitted from the same side of the object on which light illuminates the object. This type of illumination is known primarily as epi-illumination. In epi-illumination the light emission from an object may be in the form of reflection, in which case the illumination light is modulated upon reflection from the object, or it may be in the form of fluorescence, in which case the illumination light induces fluorescent emission by the object at a different wavelength from the illumination light, as determined by the fluorescence characteristics of the object. The latter case is known as epi-fluorescence.
Several different types of illumination may be used in epi-illumination microscopy. Perhaps most common is Kohler illumination, where a light source is imaged by an illumination lens, usually referred to as a condenser, into the pupil of an imaging lens, thereby providing uniform illumination of the object. The light source is ordinarily disposed actually or virtually on the optical axis of the imaging lens. This is typically accomplished by placing a beam splitter between the imaging lens and the image plane so as to change the direction of propagation of illumination light from lateral to axial, while permitting the image light to propagate axially to the image plane.
Another type of illumination that is sometimes used with epi-illumination microscopy is critical illumination. In this case, the light source is imaged at the object plane. This provides a shorter illumination system, but requires that the light source provide uniform radiance. Like Kohler illumination, the light source is ordinarily disposed actually or virtually on the optical axis of the imaging lens.
A third type of illumination that is often used with epi-illumination microscopy is dark-field, or “anti-specular,” illumination. In this case, the illumination light is directed toward the object from a location sufficiently far off the optical axis of the imaging lens that light that is specularly reflected from the object does not enter the entrance pupil of the imaging lens. In the absence of an object, no illumination light is collected by the imaging lens. In the presence of an object, light scattered by the object is collected and imaged by the imaging lens.
In the foregoing it is assumed that the entire field of view of the imaging lens is simultaneously imaged. However, in a confocal microscope only one point in object space is imaged. This is accomplished by placing a “pinhole” stop at the image plane of the microscope matched to a point source in the object plane and scanning the object laterally, either by moving the object or the microscope, or moving the scanning the beam through the microscope using, for example, scan mirrors. The light passed by the pinhole is detected and related to relative object position as the scan occurs and the output of the detector is used to produce an image of the object as a whole. In this case, light from the light source is focused to the point on the object plane that is currently imaged. This is typically accomplished by placing a beam splitter between the imaging lens and the image plane so as to pass image light to the image plane while reflecting source light from a virtual image plane created by the beam splitter along the optical axis of the microscope toward the object plane.
The recent development of array microscopes, also known as miniaturized microscope arrays, presents new challenges for illumination. In array microscopes a plurality of laterally-distributed optical imaging elements having respective longitudinal optical axes are configured to image respective sections of an object and disposed with respect to an object plane in front of the imaging elements so as to produce at respective image planes respective images of the respective sections of the object in back of the imaging elements. The individual lenses of this array are formed of small optical elements, or “lenslets,” that place severe constraints on providing illumination. Indeed, the multiplicity of lenslets arranged in an array and the small dimensions of the array suggest that prior art epi-illumination techniques cannot be used. Yet, a principal application for array microscopes is to image specimens, such as biological microarrays for protein analysis that are sufficiently opaque that dia-illumination cannot be used effectively.
Accordingly, there is an unfulfilled need for methods and devices for providing epi-illumination of objects to be imaged by array microscopes using epi-illumination.